Progesterone is required to maintain pregnancy and low progesterone concentrations are associated with infertility. Blood progesterone concentrations are influenced by rates of secretion and metabolism/clearance. There is evidence that modern dairy cows maintain lower blood progesterone concentrations than those measured in cattle several decades ago (Lucy et al. (1998) “Reproductive endocrinology of lactating dairy cows selected for increased milk production,” J. Anim. Sci., 76 (Suppl. 1):296). Larger corpora lutea secrete more progesterone and have a positive effect on pregnancy recognition and pregnancy rates, but there is evidence that dairy cows have smaller than desirable corpora lutea in some circumstances (Lucy 2001, supra; Vasconcelos et al. (2001) “Reduction in size of the ovulatory follicle reduces subsequent luteal size and pregnancy rate,” Theriogenology, 56:307-314). The liver is the primary site of progesterone metabolism. Recent studies show that increased feed intake increases liver blood flow and increases the rate of progesterone clearance, thus decreasing serum progesterone concentrations (Sangsritavong et al. (2000) “Liver blood flow and steroid metabolism are increased by both acute feeding and hypertrophy of the digestive tract,” J. Anim. Sci., 78 (Suppl 1)221; and Wiltbank, M. C. et al. (2001) “Novel effects of nutrition on reproduction in lactating dairy cows,” J. Dairy Sci., 84 (Suppl. 1):84).
Low serum progesterone during the luteal phase of the estrus cycle would be associated with low first service conception rate. Low progesterone concentrations may result from inadequate secretion, or alternatively high levels of metabolism/clearance, even when insemination has produced a potentially viable embryo. Low progesterone would allow the generation of prostaglandin by uterine endometrium at around day 16 of the bovine estrus cycle, resulting in luteolysis and induction of ovulation, thus embryonic death and failure to maintain the pregnancy (Binelli, M. et al. (2001) “Antiluteolytic strategies to improve fertility in cattle,” Theriogenology, 56:1451-1463). Increasing serum progesterone or maintaining the proper levels of serum progesterone in fertilized animals is a promising therapeutic method for maintaining pregnancy and preventing pregnancy loss.
Currently, several hormone therapies are used to increase fertility or to maintain pregnancy. Thatcher et al. (2001 Theriogenology 55:75-89) describes the effects of hormonal treatments on the reproductive performance of cattle. Hormonal treatments include administration of bovine somatotrophin (bST) and human chorionic gonadotropin (hCG). D'Occhio et al. (2000 Anim. Reprod. Sci. 60-61:433-442) describes various strategies for beef cattle management using gonadotropin releasing hormone (GnRH) agonist implants. De Rensis et al. (2002 Theriogenology 58(9):1675-1687) describes the effect on dairy cows of administering GnRH or hCG before artificial insemination. Martinez et al. (1999 Anim. Reprod. Sci. 57:23-33) describes the ability of porcine luteinizing hormone (LH) and GnRH to induce follicular wave emergence in beef heifers on Days 3, 6, and 9 of the estrus cycle, after ovulation (Day 0), without insemination. Santos et al. (2001 J. Animal Science 79:2881-2894) describes the effect on reproductive performance of intramuscular administration of 3,300 IU of hCG to high-producing dairy cows on Day 5 after artificial insemination. Lee et al. (1983 Am. J. Vet. Res. 44(11):2160-2163) describes the effect on dairy cows of administering GnRH at the time of artificial insemination. U.S. Pat. Nos. 5,792,785 (issued Aug. 11, 1998) and 6,403,631 (issued Jun. 11, 2002) describe methods and compositions for administering melatonin before and after insemination to enhance pregnancy success in an animal. Chagas e Silva et al. (2002 Theriogenology 58(1):51-59) describes plasma progesterone profiles following embryo transfer in dairy cattle. Weems et al. (1998 Prostaglandins and other Lipid Mediators) describes the effects of hormones on the secretion of progesterone by corpora lutea (CL) from non-pregnant and pregnant cows. U.S. Pat. No. 4,780,451 (issued Oct. 25, 1988) describes compositions and methods using LH and follicle stimulating hormone to produce superovulation in cattle; Farin et al. (1988 Biol. Reprod. 38:413-421) describes the effect on ovine luteal weight of intravenous administration of 300 IU of hCG on Days 5 and 7.5 of the estrus cycle, without insemination. Hoyer and Niswender (1985 Can. J. Physiol. Pharmacol. 63(3):240-248) describe the regulation of steroidogenesis in ovine luteal cells. Juengel and Niswender (1999 J. Reprod. Fertil. Suppl. 54:193-205) describe the molecular regulation of luteal progesterone in domestic ruminants. U.S. Pat. No. 5,589,457 (issued Dec. 31, 1996) describes methods for synchronizing ovulation in cattle using GnRH, LH, and/or hCG and PGF2α.
Many of these treatments use hormones or hormone analogs from the glycoprotein hormone family, which consists of the pituitary proteins luteinizing hormone (LH), follicle-stimulating hormone (FSH), thyroid stimulating hormone (TSH) and chorionic gonadotropin (CG). The gonadotropins, which include CG, FSH and LH, are essential for reproductive function. They are heterodimers composed of two non-covalently associated α and β subunits. Both subunits are glycosylated, containing asparagine (N)-linked oligosaccharides and, in the case of the CGβ subunit, O-linked carbohydrates are also present in a cluster of amino acids at the C-terminus. The individual human β subunits are encoded by separate genes, and the LHβ and CGβ proteins are structurally and functionally similar; having more than 80% amino acid identity (Pierce J G, Parsons (1981) “Glycoprotein hormones: structure and function,” Biochem. 50:465-495). Within a species, the α subunit amino acid sequence is common to all four hormones (Pierce J G, Parsons (1981) Biochem. 50:465-495).
All mammals synthesize LH, but CG has only been identified in primates and equids. In contrast to the primates, the equine LHβ and equine CGβ proteins are encoded by the same gene and have the same protein sequence. However, the N-linked oligosaccharides in placental equine CG (eCG) contain terminal galactose and sialic acid, while GalNAc sulfate is the primary terminal residue in pituitary equine LH (eLH). The carbohydrate content of eCG exceeds 40% of its mass and is the most glycosylated of the entire species of glycoprotein hormones (Bousfield et al. (2001) “Identification of twelve O-glycosylation sites in equine chorionic gonadotropin and equine luteinizing hormone β by solid-phase Edman degradation,” Biol Reprod. 64:136-147; Moore and Ward (1980) “Pregnant mare serum gonadotropin. An in vitro biological characterization of the lutropin-follitropin dual activity,” J Biol Chem 255: 6923-6929). This is attributed to a greater abundance of O-linked carbohydrate units compared with the primate CG. By contrast, the carbohydrate content of eLH is 25% (Bousfield et al. (2001) Biol Reprod. 64:136-147).
In the mare and stallion, use of a variety of hormones from other species is unsatisfactory due to their potential in eliciting a strong immune response. One such hormone that induces an antibody response is hCG (Roser et al., 1979). As alternatives, GnRH, equine pituitary extracts and equine chorionic gonadotropin (eCG; formally called pregnant mare serum gonadotropin (PMSG)) have been tried. Short acting Buserelin (a GnRH agonist) has been successful in inducing ovulation but requires more than one injection to induce ovulation within a 48 hour period. Cystorelin (native GnRH) is also short acting requiring more than one injection and is not licensed for use in the horse. Deslorelin (a GnRH analog) works very well in stimulating ovulation within 24-48 hours. Deslorelin has been on the market in two forms; a slow release implant or a slow release injectable. The implant, Ovuplant™, was found to be effective in inducing ovulation within 48 hours but in some mares prevented them from coming back into heat for several weeks when applied as directed (Johnson et al., 2002). Ovuplant™ is currently off the market. The injectable form of GnRH works well and is somewhat available as a “compounding reagent”.
Pituitary extracts can be effective reproductive therapeutics but contain contaminants and may vary in their amounts of LH and FSH. Treatment with pituitary extracts or GnRH to a mare or stallion results in exposure of the gonads to a relatively fixed ratio of LH and FSH, offering limited possibilities of manipulating the gonads for follicular development, ovulation or spermatogenesis. In addition equine pituitary extracts contain only 8-10% LH and 6-8% FSH (Guillou and Combarnous, 1983), requiring the use of large treatment doses to be effective. Equine pituitary extracts appears to increase the number of follicles for ovulation, but the number of ovulations and number of embryos obtained do not appear to always correlate with the number of follicles developed (Scoggins et al., 2002). Equine CG has been shown to have little, if any, effect in the mare to stimulate follicular development and ovulation during estrus probably due to its inability to bind to ovarian tissue during estrus (Stewart and Allen, 1979).
In order to use equine gonadotropins to improve reproduction efficiency in equines and other species, the availability of purified proteins is essential. Currently, the sources for equine gonadotropins are serum (eCG-PMSG) and whole pituitary extracts. To obtain sufficient quantities of these native hormones for such work is expensive and difficult. Preparations of pure pituitary equine gonadotropins without cross-contamination are not readily available. Given the problem of animal-to-animal variation of native equine gonadotropins and the charge heterogeneity in the N-linked carbohydrates, the ability to generate the corresponding recombinant proteins will yield gonadotropins of a more homogeneous composition that can be standardized with respect to mass and bioactivity. Such proteins will be critical for calibrating clinical laboratory assays and for breeding management, such as shortening the time to ovulation in transitional and cycling mares for natural breeding and artificial insemination. The use of recombinant forms, as opposed to hormones extracted from serum and pituitary tissue, would avoid the co-contamination of pathogens and agents with a propensity to cause prion related diseases.
There is a need in the art for improved safe therapeutics for increasing the efficiency of breeding in horses and cattle, primarily by increasing ovulation, and then by maintaining pregnancy of post-inseminated mares and cows.